In this study, three periodontal examinations were performed in SPT patients: the first at baseline, the second examination one year after four SPTs, and the third two years after eight SPTs (Fig. 1). The periodontal examination included clinical parameters (PlI, PD, CAL, GI, and BOP) and a biochemical parameter (Hb levels in the GCF). In particular, Hb, indicating a history of bleeding, has been identified in GCF [12, 13], and we demonstrated that Hb in GCF is an important marker of the progression of periodontitis [14,15,16].

When the results of the first periodontal examination were compared with the results of the second examination one year after four SPTs, all other clinical and biochemical parameter values, except the BOP value, were similar to those of the first examination. Furthermore, when comparing the results of the first periodontal disease test with the results of the third periodontal disease test two years after the eight SPTs, the PD, CAL, and GI, but not PlI and BOP, and Hb values decreased (Table 2). Not only from the first examination to the second examination, but also from the first examination to the third examination, these decreases in the PD, CAL, GI, and Hb values suggest that SPT tends to improve periodontal disease to some extent. However, from the second to the third examinations, PD and Hb showed a decreasing trend, whereas other parameter values showed an increasing trend. This suggests that periodontal disease is markedly improved one year after the first periodontal examination, but there is not much change one year after the second periodontal examination.

These six parameters correlated with each other at each time point, not only at the first and second periodontal examinations, but also at the third examination (Table 3a-c). This suggests that the values of these parameters reflect the state of periodontal tissue to some extent.

Claffey et al. [22] have already discussed the condition of periodontal tissues in SPT patients by dividing them into groups with PD values ≤ 4 mm and ≥ 5 mm. In the present study as well, clinical parameters and Hb test values were categorized into two groups based on periodontal pocket depth (PD ≤ 4 mm and PD ≥ 5 mm), and each parameter was analyzed from the first periodontal examination to the second and third periodontal examinations (Fig. 1). These changes were then analyzed (Tables 4, 5).

In the PD ≤ 4 mm group (Table 4), of the five clinical parameters, only the value of PD decreased from the first examination to the second and third examinations. In addition, Hb levels in GCF, evidence of bleeding, were also significantly decreased. In contrast, from the second to the third examinations, all parameter values, except for PD, showed an increasing tendency. These results suggest that the inflamed periodontal tissues in the PD ≤ 4 mm group did not show any significant improvement even one year after the second examination.

However, in the PD ≥ 5 mm group (Table 5), all clinical parameters and Hb values, except for the BOP value, showed significant reductions from the first examination to the second examination. Furthermore, from the first examination to the third examination, the values of these parameters, except for BOP, also decreased. In addition, comparing these parameter values between the second and third examinations, the PD and Hb values decreased from the second examination to the third examination. This reduction in the Hb value was noticeable and significant. In the PD ≥ 5 mm group, unlike the PD ≤ 4 mm group, both PD and Hb values decreased sequentially and continuously from the first to the second and third examinations.

To summarize these findings, in unclassified pocket sites, the SPT conducted for one year between the first and second periodontal examinations decreased Hb values and most clinical parameter values, except for BOP. However, from the second to the third examinations, the PD ≥ 5 mm group, which had advanced periodontal disease, showed a significant decrease in PD and Hb values, unlike the unclassified group and the PD ≤ 4 mm group. These results suggest that periodontal tissues with deep pockets that are inflamed are more likely to improve through SPT than tissues with shallow pockets that are inflamed.

Badersten et al. reported that the rate of pocket reduction after scaling and root planing depends on the depth of the periodontal pocket [24, 25]. They suggested that the deeper the periodontal pockets, the greater the reduction rate of the pockets. The results of the present study were consistent with their results in that clinical parameters decreased, and pocket improvement was observed in deep pockets with a PD of 5 mm or greater after SPT intervention during the observation period.

In contrast, the changes in BOP test results from the first periodontal examination to the second and third examinations were different from the changes in other parameters. Lang et al. already reported that the negative predictive value of the BOP test is low [5]. We have reported a large number of negative results of the BOP test in severely inflamed periodontal tissue and classified the periodontal pockets by cut-off values for BOP and PD [7]. This phenomenon, peculiar to BOP, is thought to occur because the BOP test differs from other parameter tests in that it is a diagnostic method in which an external force is applied artificially to the periodontal tissue, and the presence or absence of bleeding is observed. In contrast, in the past, we have also reported that many periodontal tissues diagnosed BOP (-) are mildly damaged due to the presence of Hb in the GCF, despite being clinically stable [14, 15]. The results of the present study and the previous study [16] with long-term follow-up showed that changes in the Hb value in GCF correlated well with changes in clinical parameter values. However, the BOP test results were found to sometimes show no correlation with the results of other clinical parameter tests and the Hb test in GCF.

BOP and PD examinations using probes are tests that can evaluate the progression of periodontal disease by observing the vulnerability of periodontal tissues and attachment loss. The Hb test in GCF measures minute amounts of hemoglobin molecules in the blood within the periodontal pocket and can detect vascular damage in periodontal tissues caused by inflammation. Based on these findings, we believe that testing for Hb in GCF may be more sensitive in detecting the early stage of periodontal inflammation than testing for BOP. Therefore, we believe that the discrepancy in the results of the two tests is not only due to differences in testing methods, but also because the detection sensitivity of each test differs depending on the stage of progression of the periodontal disease being tested.

The usefulness of various markers in GCF component analysis has been reported, and clinical applications are expected [26]. Takeuchi et al. suggested that measuring the amounts of cytokines in GCF could more accurately reflect the condition of periodontal tissues, and that SPT could reduce cytokine levels [27]. Clinical application of a simple aspartate aminotransferase kit has also been reported [28]. Our developed hemoglobin test using the IC method is highly sensitive, simple, and rapid, with test results available in 15 min, making it suitable for chairside care.

Based on the above results, adding the Hb test in GCF to clinical parameter tests, such as the BOP test, will allow for a more accurate understanding of the state of inflamed periodontal tissues. We believe that the Hb test will contribute substantially to improving the accuracy of periodontal disease assessments after treatment.